Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli
In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at...
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| Format: | Article |
| Language: | en_US |
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Springer-Verlag
2013
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| Online Access: | http://hdl.handle.net/1721.1/82623 https://orcid.org/0000-0003-0437-3157 https://orcid.org/0000-0001-6909-4568 |
| _version_ | 1826206215940603904 |
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| author | Fischer, Curt R. Tseng, Hsien-Chung Tai, Mitchell Stephanopoulos, Gregory Prather, Kristala L. Jones |
| author2 | Massachusetts Institute of Technology. Department of Chemical Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Chemical Engineering Fischer, Curt R. Tseng, Hsien-Chung Tai, Mitchell Stephanopoulos, Gregory Prather, Kristala L. Jones |
| author_sort | Fischer, Curt R. |
| collection | MIT |
| description | In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at much lower titers and yields. To pinpoint deficient steps in the recombinant pathway, we developed an analytical procedure for the determination of intracellular pools of key pathway intermediates and applied the technique to the analysis of three sets of E. coli strains expressing various combinations of butyrate biosynthesis genes. Low expression levels of the hbd-encoded S-3-hydroxybutyryl-CoA dehydrogenase were insufficient to convert acetyl-CoA to 3-hydroxybutyryl-CoA, indicating that hbd was a rate-limiting step in the production of butyryl-CoA. Increasing hbd expression alleviated this bottleneck, but in resulting strains, our pool size measurements and thermodynamic analysis showed that the reaction step catalyzed by the bcd-encoded butyryl-CoA dehydrogenase was rate-limiting. E. coli strains expressing both hbd and ptb-buk produced crotonic acid as a byproduct, but this byproduct was not observed with expression of related genes from non-clostridial organisms. Our thermodynamic interpretation of pool size measurements is applicable to the analysis of other metabolic pathways. |
| first_indexed | 2024-09-23T13:25:55Z |
| format | Article |
| id | mit-1721.1/82623 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T13:25:55Z |
| publishDate | 2013 |
| publisher | Springer-Verlag |
| record_format | dspace |
| spelling | mit-1721.1/826232022-09-28T14:07:54Z Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli Fischer, Curt R. Tseng, Hsien-Chung Tai, Mitchell Stephanopoulos, Gregory Prather, Kristala L. Jones Massachusetts Institute of Technology. Department of Chemical Engineering Fischer, Curt R. Tseng, Hsien-Chung Tai, Mitchell Prather, Kristala L. Jones Stephanopoulos, Gregory In clostridia, n-butanol production from carbohydrates at yields of up to 76% of the theoretical maximum and at titers of up to 13 g/L has been reported. However, in Escherichia coli, several groups have reported butyric acid or butanol production from recombinant expression of clostridial genes, at much lower titers and yields. To pinpoint deficient steps in the recombinant pathway, we developed an analytical procedure for the determination of intracellular pools of key pathway intermediates and applied the technique to the analysis of three sets of E. coli strains expressing various combinations of butyrate biosynthesis genes. Low expression levels of the hbd-encoded S-3-hydroxybutyryl-CoA dehydrogenase were insufficient to convert acetyl-CoA to 3-hydroxybutyryl-CoA, indicating that hbd was a rate-limiting step in the production of butyryl-CoA. Increasing hbd expression alleviated this bottleneck, but in resulting strains, our pool size measurements and thermodynamic analysis showed that the reaction step catalyzed by the bcd-encoded butyryl-CoA dehydrogenase was rate-limiting. E. coli strains expressing both hbd and ptb-buk produced crotonic acid as a byproduct, but this byproduct was not observed with expression of related genes from non-clostridial organisms. Our thermodynamic interpretation of pool size measurements is applicable to the analysis of other metabolic pathways. DuPont MIT Alliance National Science Foundation (U.S.) MIT Energy Initiative Shell Global Solutions (U.S.) National Institutes of Health (U.S.) (Biotechnology Training Program Award) 2013-12-02T19:10:46Z 2013-12-02T19:10:46Z 2010-07 2010-06 Article http://purl.org/eprint/type/JournalArticle 0175-7598 1432-0614 http://hdl.handle.net/1721.1/82623 Fischer, Curt R., Hsien-Chung Tseng, Mitchell Tai, Kristala L. J. Prather, and Gregory Stephanopoulos. “Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli.” Applied Microbiology and Biotechnology 88, no. 1 (September 13, 2010): 265-275. https://orcid.org/0000-0003-0437-3157 https://orcid.org/0000-0001-6909-4568 en_US http://dx.doi.org/10.1007/s00253-010-2749-2 Applied Microbiology and Biotechnology http://creativecommons.org/licenses/by/3.0/ application/pdf Springer-Verlag PMC |
| spellingShingle | Fischer, Curt R. Tseng, Hsien-Chung Tai, Mitchell Stephanopoulos, Gregory Prather, Kristala L. Jones Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli |
| title | Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli |
| title_full | Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli |
| title_fullStr | Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli |
| title_full_unstemmed | Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli |
| title_short | Assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant Escherichia coli |
| title_sort | assessment of heterologous butyrate and butanol pathway activity by measurement of intracellular pathway intermediates in recombinant escherichia coli |
| url | http://hdl.handle.net/1721.1/82623 https://orcid.org/0000-0003-0437-3157 https://orcid.org/0000-0001-6909-4568 |
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